Deregulation of the mechanisms controlling cell-cycle progression is a hallmark of neoplasia. Cyclin-dependent kinases (CDKs) constitute a family of well conserved serine/threonine protein kinases which are active in complexes with their regulatory subunits, the cyclins. The human genome encodes 13 CDKs, 48 CDK related kinases and 25 cyclins. The different members of the CDK family have been implicated in a range of key cellular processes: CDK1, 2, 3, 4, 6, and 7 regulate the cell cycle, CDK7, 8, and 9 interact directly with transcription factors, CDK5 and 11 control neuronal functions, CDK2, 5, 6, and 9 cell differentiation and CDK1, 2, 4, 5, 6, and 11 affect apoptosis (Knockaert et al.: J. Biol. Chem. 277: 25493-25501, 2002). CDK functions are regulated by cyclins, CDK inhibitory proteins and subcellular localization. Since many CDKs are critical regulators of cell division, the pharmaceutical industry has been targeting the discovery and development of pharmacological CDK inhibitors (CDKIs) as potential anticancer drugs. CDKIs are a diverse and heterogeneous family of small, flat heterocycles-purines, pyrimidines, flavonoids or bis-indoles that bind to the ATP binding pocket of their target CDK where they can compete with the ATP (de Azevedo et al., Eur. J. Biochem. 243:518-526, 1997).
The key regulators of the cycle are represented by 3 groups of proteins: cyclins, cyclin-dependent kinases (CDK) and cyclin-dependent kinase inhibitors (CDKI).
The earliest significant CDKIs were the CDK oligo-specific Olomoucine and the CDK pan-specific Flavopiridol (Losiewicz et al., Biochem. Biophys. Res. Commun. 201(2):589-95, 1994). Olomoucine belongs to C2, N6, N9-substituted adenines that show high efficiency and selectivity towards some CDKs; Olomoucine specifically inhibits CDK2, CDK5, and to a lesser extent Erk1 (Veselý et al., Eur. J. Biochem. 224: 771-786, 1994). With the specific objective of attaining enhanced inhibition of CDKs, Olomoucine was subjected to structural modifications. A classical structure-activity relationship study directed at modifying Olomoucine side chains generated two exceptionally potent CDKIs: Roscovitine (Seliciclib®, CYC202, Cyclacel Pharmaceuticals, Berkeley Heights, N.J.) and most recently Olomoucine II (Havlí{hacek over (c)}ek et al. 1997. J. Med. Chem. 40: 408-412; Kry{hacek over (s)}tof et al. Bioorg Med Chem Lett 12: 3283-3286, 2002).
The increased potency of Roscovitine over Olomoucine is caused by the introduction of branched C2-side chain and more bulky N9-isopropyl moiety. These changes markedly increased the complementarity of the inhibitor to the ATP-binding site of CDK2, as demonstrated by X-ray crystallography (de Azevedo et al. Eur. J. Biochem. 243:518-526, 1997). Both structural changes did not alter the selectivity of Roscovitine, but increased the cellular potency of Roscovitine 10-fold towards CDK1 and CDK2, and 20-fold towards CDK5. Olomoucine II differs from Roscovitine in having an additional ortho-hydroxyl group on the benzyl ring, yet this single modification is associated with a 10-fold higher inhibitory activity for CDK9 (Kry{hacek over (s)}tof et al., Cell Mol Life Sci. 62: 1763-1771, 2005). This increased affinity of Olomoucine II towards CDK9 is responsible for its enhanced effect on intracellular functional activities, when compared to Roscovitine. For example, Olomoucine II induces the nuclear accumulation and transcriptional activation of the tumor suppressor protein p53 at 2-3-fold lower concentrations than Roscovitine. At higher concentrations both inhibitors may inhibit some RNA synthesis by attenuating CTD domain phosphorylation of RNA polymerase II (Diwan et al., J Virol. 78: 9352-65, 2004).
The finding that Roscovitine possessed antiviral activity stimulated research aimed at targeting cellular functions, exemplified by CDKs, required for virus replication (Bresnahan et al., J. Gen. Virol. 78:1993-7, 1997; Schang et al., J Virol. 73:2161-72, 1999). Roscovitine, and other CDKIs, have the ability to inhibit replication of broad range of viruses, even in non-dividing cells and including agents that do not require cell cycle progression (Schang et al., Antivir. Chem. Chemother. 17:293-320, 2006). Human cytomegalovirus (HCMV), herpes simplex types 1 and 2 (HSV-1 & HSV-2), varicella zoster virus (VZV), Epstein Barr virus (EBV), human adenovirus (HAdV), human immunodeficiency virus (HIV), and human T cell leukemia virus (HTLV-1) are all susceptible to Roscovitine. Antiviral activities of CDK inhibitors correlate with the extent of CDK inhibition, rather than the core structure of the inhibitor (Schang et al., Antivir. Chem. Chemother. 17:293-320, 2006).
Although CDKIs were shown to inhibit multiple stages of viral replication, including splicing, DNA replication, reactivation from latency, activation of cellular or viral enzymes and intracellular trafficking, it is recognized that the antiviral activity of CDKIs is primarily mediated by inhibiting virus-encoded transcription (Kapasi and Spector, J. Virol. 82(1):394-407, 2008). Interestingly, Roscovitine can prevent initiation of viral transcription that is specific to viral genomes and independent of promoter elements (Diwan et al., J. Virol. 78: 9352-65, 2004). While Flavopiridol is CDK panspecific and inhibits transcription of most cellular and viral genes, Roscovitine is CDK oligospecific and appears not to inhibit the cellular transcription. The selectivity of Roscovitine in suppressing viral and plasmid encoded gene expression resembles an activity described for interferon alpha (Nicholl and Preston, J. Virol. 70: 6336-9, 1996). In this context it is interesting to note that interferon alpha also inhibits CDKs and promotes cell cycle arrest (Mandal et al., Oncogene 16:217-225, 1998).
The mechanisms by which CDKIs suppress virus replication have not been fully defined and can be expected to differ between agents. Consequently, it was extremely important to compare and contrast the potential antiviral properties of Roscovitine and its analogues empirically. Our previous studies had determined that Olomoucine II and many other compounds were more potent inhibitors of CDK activities than Roscovitine (Kry{hacek over (s)}tof et al., Cell Mol Life Sci. 62: 1763-1771, 2005).